Grainne Hurley

Mechanism of protection of transepithelial barrier function by Lactobacillus salivarius: strain dependence and attenuation by bacteriocin production

Enhanced barrier function is one mechanism whereby commensals and probiotic bacteria limit translocation of foreign antigens or pathogens in the gut. However, barrier protection is not exhibited by all probiotic or commensals and the strain-specific molecules involved remain to be clarified. We evaluated the effects of 33 individual Lactobacillus salivarius strains on the hydrogen peroxide (H(2)O(2))-induced barrier impairment in human epithelial Caco-2 cells. These strains showed markedly different effects on H(2)O(2)-induced reduction in transepithelial resistance (TER). The effective strains such as UCC118 and CCUG38008 attenuated H(2)O(2)-induced disassembly and relocalization of tight junction proteins, but the ineffective strain AH43324 did not. Strains UCC118 and CCUG38008 induced phosphorylation of extracellular signal-regulated kinase (ERK) in Caco-2 cells, and the ERK inhibitor U0126 attenuated the barrier-protecting effect of these strains. In contrast, the AH43324 strain induced phosphorylation of Akt and p38, which was associated with an absence of a protective effect. Global transcriptome analysis of UCC118 and AH43324 revealed that some genes in a bacteriocin gene cluster were upregulated in AH43324 under TER assay conditions. A bacteriocin-negative UCC118 mutant displayed significantly greater suppressive effect on H(2)O(2)-induced reduction in TER compared with wild-type UCC118. The wild-type strain augmented H(2)O(2)-induced phosphorylation of Akt and p38, whereas a bacteriocin-negative UCC118 mutant did not. These observations indicate that L. salivarius strains are widely divergent in their capacity for barrier protection, and this is underpinned by differences in the activation of intracellular signaling pathways. Furthermore, bacteriocin production appears to have an attenuating influence on lactobacillus-mediated barrier protection.

Natural killer cells protect mice from DSS-induced colitis by regulating neutrophil function via the NKG2A receptor

Natural killer (NK) cells are traditionally considered in the context of tumor surveillance and infection defense but their role in chronic inflammatory disorders such as inflammatory bowel disease is less clear. Here, we investigated the role of NK cells in dextran sodium sulfate (DSS)-induced colitis in mice. Depletion of NK cells impairs the survival of mice with colitis and is linked with dramatic increases in colonic damage, leukocyte infiltration, and pro-inflammatory profiles. Mice depleted of NK cells had increased numbers of neutrophils in colons and mesenteric lymph nodes, compared with control mice, in addition to acquiring a hyper-activation status. In vitro and in vivo studies demonstrate that NK cells downregulate pro-inflammatory functions of activated neutrophils, including reactive oxygen species and cytokine production, by direct cell-to-cell contact involving the NK cell–inhibitory receptor NKG2A. Our results indicate an immunoregulatory mechanism of action of NK cells attenuating DSS-induced colitis neutrophil-mediated inflammation and tissue injury via NKG2A-dependent mechanisms.

Natural Killer Cells Protect against Mucosal and Systemic Infection with the Enteric Pathogen Citrobacter rodentium

ABSTRACT Natural killer (NK) cells are traditionally considered in the context of tumor surveillance and viral defense, but their role in bacterial infections, particularly those caused by enteric pathogens, is less clear. C57BL/6 mice were orally gavaged with Citrobacter rodentium, a murine pathogen related to human diarrheagenic Escherichia coli. We used polyclonal anti-asialo GM1 antibody to actively deplete NK cells in vivo. Bioluminescent imaging and direct counts were used to follow infection. Flow cytometry and immunofluorescence microscopy were used to analyze immune responses. During C. rodentium infection, NK cells were recruited to mucosal tissues, where they expressed a diversity of immune-modulatory factors. Depletion of NK cells led to higher bacterial loads but less severe colonic inflammation, associated with reduced immune cell recruitment and lower cytokine levels. NK cell-depleted mice also developed disseminated systemic infection, unlike control infected mice. NK cells were also cytotoxic to C. rodentium in vitro.

The Sphingosine-1-Phosphate Analogue FTY720 Impairs Mucosal Immunity and Clearance of the Enteric Pathogen Citrobacter rodentium

ABSTRACT The sphingosine-1-phosphate (S1P) analogue FTY720 is therapeutically efficacious in multiple sclerosis and in the prevention of transplant rejection. It prevents the migration of lymphocytes to sites of pathology by trapping them within the peripheral lymph nodes, mesenteric lymph nodes (MLNs), and Peyers patches. However, evidence suggests that its clinical use may increase the risk of mucosal infections. We investigated the impact of FTY720 treatment on susceptibility to gastrointestinal infection with the mouse enteric pathogen Citrobacter rodentium. This attaching and effacing bacterium induces a transient bacterial colitis in immunocompetent mice that resembles human infection with pathogenic Escherichia coli. FTY720 treatment induced peripheral blood lymphopenia, trapped lymphocytes in the MLNs, and prevented the clearance of bacteria when mice were infected with luciferase-tagged C. rodentium. FTY720-treated C. rodentium-infected mice had enhanced colonic inflammation, with significantly higher colon mass, colon histopathology, and neutrophil infiltration than vehicle-infected animals. In addition, FTY720-treated infected mice had significantly lower numbers of colonic dendritic cells, macrophages, and T cells. Gene expression analysis demonstrated that FTY720-treated infected mice had an impaired innate immune response and a blunted mucosal adaptive immune response, including Th1 cytokines. The data demonstrate that the S1P analogue FTY720 adversely affects the immune response to and clearance of C. rodentium.

409 The Colonic Adherent-Invasive Escherichia coli Strain HM605 Induces Anti-Apoptotic Responses in Intestinal Epithelial Cells, Reduces Barrier Integrity and Worsens Experimental Colitis

G A A b st ra ct s (MMPs) are zinc-dependent neutral endopeptidases that participate in degradation of extracellular matrix proteins involved in a variety of pathological processes including inflammation and cancer. MMP9 protein expression and activity is undetectable in most healthy adult tissues including the intestine but is highly expressed in inflammatory state. We have shown that despite being a mediator of acute inflammation MMP9 plays a protective role in the development of CAC. Aim of the present study is to determine the necessity and sufficiency of epithelial derived MMP9 mediated p53 activation via Notch1 signaling for its tumor suppressive function in CAC, by using MMP9 transgenic mice (Tg-villin-MMP9) that specifically overexpresses MMP9 in the colonic epithelium. Methods: Age and gender matched Tg-villin-MMP9 and their wild type littermates (WT) mice were used for in vivo experiments and embryonic fibroblasts (MEFs) isolated from WT and MMP9-/mice were used for in vitro experiments. Mice were exposed to dextran sulfate sodium (DSS, 3% in drinking water) and were given 3 DSS cycles of one week each and with two weeks apart and were sacrificed at day 75. Colons were processed for inflammation and polyps. Swiss rolls of colonic tissues were histologically examined. Western blot (WB) analysis was done with the colonic mucosal stripping. Results: Tg-villin-MMP9 mice were more resistant to CAC as evidenced by decreased tumor burden. In addition, decreased erosion of mucosal layer, infiltration of neutrophils and dysplasia were seen in the polyps of Tg-villin-MMP9 mice compared to WTs both inducedwith CAC.WB analysis indicated significantly increased protein expression of NICD (active Notch1), p53, p21WAF1/Cip1, Bax-1 and caspase-3 compared to WTs mice both induced with CAC. TUNEL staining showed increased apoptosis in the colons of Tg-villin-MMP9 mice compared to WTs mice both induced with CAC. MMP9 overexpression (by stable transfection) in MMP9-/MEFs resulted in significantly increased protein expression of NICD, p53 and p21WAF1/Cip1 expressions compared to non-transfected MMP9-/MEFs, and were comparable to WT MEFs. Conclusion: Together, the data show that constitutive expression of MMP9 in colonic epithelium modulates apoptosis and cell cycle arrest, and thereby acts as a tumor suppressor in CAC. This study elucidates the novel tumor suppressive role of MMP9 in malignant transformation of the colonic epithelium in context of chronic inflammation which may provide new diagnostic and therapeutic approaches.